The kinesin superfamily are comprised of proteins which utilize a conserved catalytic motor domain to generate intracellular movement of vesicles or macromolecules along microtubules in diverse eukaryotic cellular processes (e.g., cell proliferation). Over 90 kinesin proteins can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. The motor domain is a compact structure of approximately 340 amino acids, and can be located at the N-terminus, in the internal region, or at the C-terminus of the kinesin molecule. Most of the kinesin proteins have an N-terminal catalytic motor domains, e.g., the BimC and the KHC families (See, e.g., Goldstein et al., Annu. Rev. Cell Dev. Biol., 15:141-83, 1999; Moore, J. D. and Endow, S. A., Bioassays 18:207-219, 1996). During mitosis, kinesins organize microtubules into the bipolar structure that is the mitotic spindle, mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle associated with specific phases of mitosis. These “molecular motors” translate energy released by hydrolysis of ATP into mechanical force which drives the directional movement of cellular cargoes along microtubules.
The prototypical native kinesin molecule is a heterotetramer comprised of two heavy polypeptide chains (KHC's) and two light polypeptide chains (KLC's). The KHC homodimer is typically referred to as “kinesin” and is classified as a member of the KHC kinesin family (Goldstein et al., Annu. Rev. Cell Dev. Biol., 15:141-83, 1999). The human form of KHC has been cloned (Navone et al., J. Cell Biol., 117:1263-75 (1992)). Human KHC N-terminal fragments have reportedly been expressed in E. coli and purified (Fujiwara, et al., Biophys. J. 69:1563-8, 1995; Vale et al., Nature 380:451-3, 1996). Crystal structure of KHC motor domain has been reported (Kull et al., Nature 380:550-555, 1996). Motility activity of KHC has also been reported.
Another notable kinesin that has been identified is kinesin-like spindle protein (“KSP”), a member of the BimC kinesin family that is characterized by a conserved, globular motor domain at the amino terminus followed by a non-conserved, rod-like helical coiled-coil domain and a BimC box at the carboxyl terminus (Endow, Trends Biol. Sci. 16:221-225, 1991; Sanders et al., J. Cell Biol. 128:617-624, 1995). During mitosis, KSP associates with microtubules of the mitotic spindle. Microinjection of antibody directed against KSP into human cells prevents spindle pole separation during prometaphase, giving rise to monopolar spindles and causing mitotic arrest. KSP and related kinesins bundle antiparallel microtubules and slide them relative to one another, thus forcing the two spindle poles apart. KSP may also mediate in anaphase B spindle elongation and focusing of microtubules at the spindle pole.
Human KSP (also termed HsEg5) has been cloned and characterized (see, e.g., Blangy et al., Cell, 83:1159-69 (1995); Galgio et al., J. Cell Biol., 135:399-414, 1996; Whitehead et al., J. Cell Sci., 111:2551-2561, 1998; Kaiser, et al., J. Biol. Chem., 274:18925-31, 1999; GenBank accession numbers: X85137, NM 004523). Drosophila (Heck et al., J. Cell Biol., 123:665-79, 1993) and Xenopus (Le Guellec et al., Mol. Cell Biol., 11:3395-8, 1991) homologs of KSP have been reported. Drosophila KLP61F/KRP130 has reportedly been purified in native form (Cole, et al., J. Biol. Chem., 269:22913-22916, 1994), expressed in E. coli, (Barton, et al., Mol. Biol. Cell, 6:1563-74, 1995) and reported to have motility and ATPase activities (Cole, et al., supra; Barton, et al., supra). Xenopus Eg5 was expressed in E. coli and reported to possess motility activity (Sawin, et al., Nature, 359:540-3, 1992; Lockhart and Cross, Biochemistry, 35:2365-73, 1996; Crevel, et al, J. Mol. Biol., 273:160-170, 1997) and ATPase activity (Lockhart and Cross, supra; Crevel et al., supra).
Besides KSP, other members of the BimC family include BimC, CIN8, cut7, KIP1, KLP61F (Barton et al., Mol. Biol. Cell. 6:1563-1574, 1995; Cottingham & Hoyt, J. Cell Biol. 138:1041-1053, 1997; DeZwaan et al., J. Cell Biol. 138:1023-1040, 1997; Gaglio et al., J. Cell Biol. 135:399-414, 1996; Geiser et al., Mol. Biol. Cell 8:1035-1050, 1997; Heck et al., J. Cell Biol. 123:665-679, 1993; Hoyt et al., J. Cell Biol. 118:109-120, 1992; Hoyt et al., Genetics 135:35-44, 1993; Huyett et al., J. Cell Sci. 111:295-301, 1998; Miller et al., Mol. Biol. Cell 9:2051-2068, 1998; Roof et al., J. Cell Biol. 118:95-108, 1992; Sanders et al., J. Cell Biol. 137:417-431, 1997; Sanders et al., Mol. Biol. Cell 8:1025-1033, 1997; Sanders et al., J. Cell Biol. 128:617-624, 1995; Sanders & Hoyt, Cell 70:451-458,1992; Sharp et al., J. Cell Biol. 144:125-138, 1999; Straight et al., J. Cell Biol. 143:687-694, 1998; Whitehead & Rattner, J. Cell Sci. 111:2551-2561, 1998; Wilson et al., J. Cell Sci. 110:451-464, 1997).